ABSTRACT Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), continues to take a severe toll on world health, and the number of drug resistant TB cases is growing. Thus, there is a significant need to develop new TB drugs that target novel pathways of Mtb. Mce transporters are mycobacterial lipid uptake pathways that function in Mtb pathogenesis. More specifically, the Mce4 transporter imports host cholesterol, which is known to be utilized by Mtb during infection. Consequently, Mce transporters are a potential target for TB drug development. However, we lack even the most basic details of how Mce transporters work. Mce transporters appear likely to be highly complex systems involving numerous components. Along with two proposed permeases, each mce operon encodes six Mce proteins that are proposed to function as solute binding proteins that bind lipid. However, no single Mce protein has been studied for a function in transport and not a single protein-protein interaction is known for Mce transporters. Adjacent to mce operons there are pairs of genes encoding Mam (Mce-associated membrane) proteins and orphaned mam (omam) genes that are unlinked from mce loci also exist. We recently identified an orphaned Mam (OmamA), which is the only Mam/Omam studied to date, as functioning in Mce transporters through a role involving stabilization of Mce proteins. OmamA and other Mam/Omam proteins may have roles analogous to that of VirB8, which functions in assembly and stabilization of Gram negative Type IV secretion systems (T4SSs), as Omam, Mam and VirB8 proteins are predicted to be structurally similar. We hypothesize that Mce transporters are large macromolecular complexes that span the cell wall, akin to T4SSs, with each of the Mce proteins having unique functions and Mam/Omam proteins being additional components that function in stabilization and/or assembly of the complex. In this developmental R21, we will use the M. smegmatis Mce4 transporter as a model system to perform a systematic analysis of the requirements of individual Mce and Mam/Omam proteins. We will also identify the first examples of functional residues and domains in these Mce transporter components and identify protein interactions and Mce transporter complexes in mycobacteria. Through establishing the core properties of Mce systems these studies will significantly advance our current understanding of these important transport pathways and improve our knowledge of lipid transport in diverse bacteria. Our results may additionally reveal strategies for inhibiting lipid uptake by Mce transporters that could serve as the basis of novel TB therapeutics.